Cell-generated mechanical stresses are critical during a number of physiological processes, including embryogenesis, cell migration, cell proliferation and tissue formation. However, there is no method currently available to measure cellular traction forces exerted during adhesion and migration in 3-D scaffolds. This information is critical for understanding the contribution of cell-cell and cell-extracellular matrix (ECM) interactions in processes such as wound healing, cancer metastasis, cardiovascular disease, and chronic inflammation. To partially address this need, a technique called Traction Force Microscopy was developed several years ago to quantify and map the traction field created by a cell on its substrate (1, 2). This technique has provided valuable information about the mechanisms of cell migration and adhesion and has demonstrated key differences in the mechanics of normal and diseased states. However, this currently used technique is limited--- the state-of-the-art is a 2-D calculation of the cell-generated forces on a planar substrate. It is well-established that 2D substrates lack the physiologically realistic environment that a 3D ECM provides cells in vivo. Therefore, to more accurately recapitulate the cellular microenvironment, these measurements need to be done in 3-D, where all sides of the cell are capable of binding to the extracellular matrix, deforming the substrate and using cell-generated traction stresses to migrate. The proposal seeks to develop and build an instrument (hardware) and a computational algorithm (software) that calculates the stresses exerted by cells embedded in 3D matrices for studies of the dynamic processes of cell adhesion and migration in ECMs which mimic the native in vivo environment. We expect that just as 2D Traction Force Microscopy has been widely implemented; our tool will be of wide-spread interest to the large community of scientists interested in mechanism of cell migration and adhesion for a number of different physiological systems and disease conditions. PUBLIC HEALTH RELEVANCE (provided by applicant): In this proposal, we plan to develop a 4D traction force microscope (hardware and software) that can measure the forces that cells exert against their substrate during adhesion and migration in 3D space and time. Such a device is essential to understand the molecular machinery that drives cell migration in physiological processes such as wound healing, tissue formation, and during the progression of diseases including cancer metastasis and atherosclerosis.